Fibrinogen, also known as clotting factor I, plays a key role in haemostasis and wound healing. It is a glycoprotein synthesized in the liver with an apparent molecular weight of 340,000 Da, is composed of two dimers, each of them built of three pairs of non-identical polypeptide chains called Aα, Bβ and γ linked by disulfide bridges. It circulates in the blood stream at a concentration of approximately 1.5-4.0 mg/ml. Upon injury of blood vessels, blood platelets are activated and a plug is formed. Fibrinogen is involved in primary haemostasis by aiding cross-linking of activated platelets.
In parallel activation of the clotting cascade is initiated. As the endpoint, fibrinogen is converted into fibrin by proteolytic release of fibrinopeptide A and—at a slower rate—fibrinopeptide B by thrombin. The soluble fibrin monomers are assembled to double stranded twisted fibrils. Subsequently these fibrils are arranged in a lateral manner, resulting in thicker fibers. These fibers are then cross-linked by FXIIIa to a fibrin network, which stabilizes the platelet plug by interactions of the fibrin with activated platelets, resulting in a stable clot.
Disorders and Deficiencies
Congenital afibrinogenaemia is a rare bleeding disorder, where patients are suffering from inadequate clotting of the blood due to lacking or malfunction of fibrinogen. This medical condition might lead to spontaneous bleeding episodes or excessive bleeding after minor traumata or during interventional procedures.
Acquired deficiencies in fibrinogen are much more common than congenital afibrinogenaemia and may be induced by haemodilution or other events such as blood losses during surgery, traumata, disseminated intravascular coagulation (DIC) or sepsis.
Fibrinogen deficiencies can be corrected to normal fibrinogen levels in plasma of about 1.5-4 g/l by replacement therapy with intravenous infusion of fresh frozen plasma or cryoprecipitate. However, these treatments are afflicted with the risk of introduction of pathogens, e.g. viruses or prions, into a patient and are thereby generating additional disorders. It is thus advisable to intravenously apply virus inactivated fibrinogen compositions to restore fibrinogen at physiological levels in a save way.
While there exists fibrinogen in preparations called fibrin glue, fibrinogen adhesive, tissue glue and similar, these preparations are intended for topical use as powders, pastes, foams or in combination with fabrics as plaster on wounds, they are not useable for intravenous application as their consistency and composition would immediately initiate thrombotic events when being injected. These preparations additionally contain thrombin, calcium salts and relatively high amounts of coagulation factor XIII. Examples for such preparations are US-A1-2008/003272, WO-A-95/22316 or US-A1-2008/181878.
Processes for fibrinogen production are known from EP-A1-1 240 200 which relates to a method of purifying fibrinogen from a fibrinogen containing solution, comprising, application of a fibrinogen containing solution to an ion exchange matrix, under conditions such that fibrinogen binds to the matrix, washing the ion exchange matrix with a buffer solution comprising at least one ω-amino acid, eluting the fibrinogen from the matrix with a buffer consisting of 10 mM Tris, 10 mM citrate, 45 mM sucrose and NaCl at a concentration of 200 mM to 1.0 M, and optionally recovering the fibrinogen from the eluate.
EP-A1-0 771 324 refers to a process for production of a virus free fibrinogen concentrate which is obtained by subjecting a solubilised plasma fraction containing fibrinogen to a chemical viral inactivation treatment, i.e. a S/D or solvent/detergent treatment, subjecting the resulting viral-inactivated fraction to precipitation in a solution containing an amino acid at an acidic pH to obtain a supernatant, filtering the supernatant to obtain a purified fibrinogen concentrate, and recovering the purified fibrinogen concentrate. The recovered fibrinogen concentrate is subjected to ultra violet radiation for a second virus inactivation. The product is stabilized and lyophilized prior to a third virus inactivation step.
EP-A1-1 519 944 teaches the use of an immobilized metal ion affinity chromatography matrix under conditions that the fibrinogen and plasminogen bind to the matrix, and selectively eluting the fibrinogen and 93% of plasminogen separately from the matrix.
EP-A1-0 555 135 discloses a method for production of an intravenously applicable fibrinogen by purification of a fibrinogen solution by means of an anion exchange gel based on cross-linked agarose comprising quaternary amine groups. The fibrinogen produced is said to be free of factor VIIIc.
EP-A1-1 457 497 refers to a process for removing viruses in fibrinogen solutions characterized by stabilization and freezing of the solution and subsequent thawing thereof. Separation of undissolved materials occurs prior to dilution of the protein and is followed by nanofiltration of the resultant solution using filters of a pore size smaller than 35 nm.
US-A1-2006/0009376 also discloses a method to manufacture fibrinogen, following repeated dissolution and precipitation of fibrinogen to remove factor XIII.
WO-A2-2009/155626 refers to a process for purification of fibrinogen by solubilisation of cryoprecipitate or Cohn-Fraction I in an EDTA-solution at a temperature range of 3° C. to 5° C., followed by fractionated precipitation in a temperature range of 2° C. to 4° C. and dissolution of the last precipitate in an EDTA-solution. Virus inactivation with S/D reagents and nanofiltration for improvement of pathogen safety occur in the presence of EDTA. Inhibition of residual amounts of proteases in the produced concentrate is accomplished by addition of AT-III, heparin-cofactor-II and C1-esterase-inhibitor.
U.S. Pat. No. 7,919,592 B2 describes a method for removing viruses from fibrinogen solutions by addition of chaotropic substances, chosen from arginine, guanidine, citrulline and urea, there salts or derivatives and subsequent filtration through nanofilters of various pore size. Goheen, S. C. et al. report in Journal of Chromatography A. 816 (1998) 89-96, about HPLC ion-exchange chromatography of the plasma proteins albumin, fibrinogen, and immunoglobulin (G) on nonporous column materials containing either quarternary amine or sulfopropyl functional groups.